BackGeneral Questions
- What is the required biosafety level for using recombinant adenovirus?
- How to check the efficiency of adenovirus infection in my target cells?
- What's the optimal concentration of viruses for infection?
- What are viral particle (VP), plaque formation unit (PFU), and infectious unit (IFU)?
- How are virus titers determined?
- For in vitro use (cell culture studies), is viral purification required?
- What are the recommended storage conditions of recombinant Adenoviruses?
- What are RCAs?
What is the required biosafety level for using recombinant adenovirus?
The recombinant adenoviruses are replication deficient due to deletions in the E1 and E3 regions. According to references issued by the NIH Office of Biosafety, recombinant human adenovirus has been classified in biosafety level II for agents considered of ordinary potential harm, and you need BL-2 level facility to work with it. It should be noted that cell culture facilities in most institutes are certified as BL-2 level.
Wild type, replication competent adenoviruses could cause cold symptoms but generally do not cause serious illness. For more information on biosafety levels please visit http://bmbl.od.nih.gov
Wild type, replication competent adenoviruses could cause cold symptoms but generally do not cause serious illness. For more information on biosafety levels please visit http://bmbl.od.nih.gov
How to check the efficiency of adenovirus infection in my target cells?
The Adenovirus has a very broad host range; it can infect human and other mammalian cell lines or primary cells, including dividing and non-dividing cells. There are in fact very few cell lines that cannot be infected. It has been shown that cells of Hematopoietic origin are more resistant to adenovirus infection, and so may need high quantities of viruses to achieve sufficient infection levels as those cells have only limited CAR receptors expressed on cell membrane.
For your convenience, we offer some marker adenoviruses, such as Ad-CMV-beta-Gal or Ad-EGFP to allow you to evaluate infection efficiency in your target cells.
For your convenience, we offer some marker adenoviruses, such as Ad-CMV-beta-Gal or Ad-EGFP to allow you to evaluate infection efficiency in your target cells.
What's the optimal concentration of viruses for infection?
The appropriate MOI (multiplicity of infection) of recombinant viruses used for infecting cells is very important for the outcome of your experiments. If MOI is low, it will not give 100% of infection. If MOI is too high, it will cause cytotoxicity or other undesired effects. You should use the minimal virus concentration that will give 100% gene delivery. This optimal concentration differs dramatically among different cell types. To determine optimal concentration of virus, you could perform pilot experiments by using marker adenoviruses, such as Ad?-Gal or AdEGFP. For most cell types, a MOI of
2 x 105 to 1 x 106 per ml of media gives 100% of infection without visible side effects. For your reference, we recommend the following amount virus-containing media for infection:
10-cm plate: 5 ml per plate
6-well plate: 1 ml per well
12-well plate: 0.5 ml per well
24-well plate: 0.2 ml per well
This volume roughly covers the surface area of each well or plate.
10-cm plate: 5 ml per plate
6-well plate: 1 ml per well
12-well plate: 0.5 ml per well
24-well plate: 0.2 ml per well
This volume roughly covers the surface area of each well or plate.
What are viral particle (VP), plaque formation unit (PFU), and infectious unit (IFU)?
Viral particles (VPs) represent the total number of viral particles (infectious and infection-deficient combined). Due to variations in virus preparations, the ratio of infectious /non-infectious varies significantly and therefore, VP does not reflect the concentration of virus in a preparation. PFU ( plaque forming unit) represents the number of infectious or live viruses. It reflects the concentration of infectious viruses in a preparation. IFU (infectious unit) is biologically equivalent to PFU. For most virus preps, the VP/PFU ratio is 20:1 to 50:1.
How are virus titers determined?
There are three commonly used protocols for determining adenovirus titer: (1)OD260 Assay, (2)Plaque Formation Assay, and (3)End-point Dilution Assay.
OD260 assay measures the concentration of viral DNA. It does not distinguish intact, infectious viruses from damaged, non-infectious ones. It is a physical assay measuring the number of total viruses, live and dead. Based on OD260, the concentration of viral particles (VP) could be obtained. To measure the OD260, the virus stock has to be purified first. On the other hand, plaque formation assay measures the concentration of infectious viruses, and therefore it is a biological assay. Basically, a monolayer of 293 cells is infected with a series of virus dilutions from 101 to 1012. Viruses will propagate in infected cells, and eventually cause complete cytotoxicity effects (CPE) in that cell, and get released. The released viruses will infect neighboring cells, and the whole process will be repeated, eventually leading to the formation of holes or plaques on the cell monolayer. In order to prevent the diffusion of viruses among plaques, a layer of agarose is laid on top of cells after initial infection. The biological principle for End-point Dilution assay is similar to the plaque formation assay, although the procedure and measurement is different, and the formula for calculating the virus titer is a bit more complicated. Although both the Plaque Formation Assay and End-point Dilution assay gives the titer of infectious or working viruses, they are scored by human eyes and subjected to human and procedure variations. For the same virus stock, it is quite common that different people will get significantly different titer readings.
OD260 assay measures the concentration of viral DNA. It does not distinguish intact, infectious viruses from damaged, non-infectious ones. It is a physical assay measuring the number of total viruses, live and dead. Based on OD260, the concentration of viral particles (VP) could be obtained. To measure the OD260, the virus stock has to be purified first. On the other hand, plaque formation assay measures the concentration of infectious viruses, and therefore it is a biological assay. Basically, a monolayer of 293 cells is infected with a series of virus dilutions from 101 to 1012. Viruses will propagate in infected cells, and eventually cause complete cytotoxicity effects (CPE) in that cell, and get released. The released viruses will infect neighboring cells, and the whole process will be repeated, eventually leading to the formation of holes or plaques on the cell monolayer. In order to prevent the diffusion of viruses among plaques, a layer of agarose is laid on top of cells after initial infection. The biological principle for End-point Dilution assay is similar to the plaque formation assay, although the procedure and measurement is different, and the formula for calculating the virus titer is a bit more complicated. Although both the Plaque Formation Assay and End-point Dilution assay gives the titer of infectious or working viruses, they are scored by human eyes and subjected to human and procedure variations. For the same virus stock, it is quite common that different people will get significantly different titer readings.
For in vitro use (cell culture studies), is viral purification required?
No. If viruses will be used in in vitro cell cultures, double CsCl purification is not required. For in vivo studies (i.e. animal studies), purification is essential in order to remove defective particles, cell debris, and small amounts of media components, since these contaminants induce significant immune responses. In addition, CsCl purification will concentrate the virus to a level suitable for in vivo injections.
What are the recommended storage conditions of recombinant Adenoviruses?
For long-term storage, the virus should be kept at -80°C, especially after CsCl or chromatography purification. At -80°C in 10 mM Tris (pH 8.0) plus 10% glycerol, the viruses could be stable for several years. Alternatively, viral stock could be stored in 10mM Tris (pH 8.0) plus 4% sucrose for in vivo injection as it is more difficult to do in vivo injection with glycerol formulation.
What are RCAs?
One concern when working with adenoviral vectors is the rare occurrence of replication competent adenoviruses (RCAs) in a population of replication-deficient viruses. RCAs can emerge as a result of the rare double crossover through overlapping sequences present in the recombinant adenovirus and the genome of 293 cells. This event results in the replacement of the transgene by E1 region. Once this happens, the adenovirus could replicate in target cells (non-permissive cells). To detect RCA, non-permissive cells, such as A549 cells, are incubated with the viral stocks and monitored for cytopathic effects (CPE). According to NIH guideline, <1 plaque in about 104 viruses is considered safe to use in clinical trials. To avoid the occurrence of RCA, viruses should be produced and amplified in low passage 293 cells.